This invention relates to specific binding members for a foetal isoform of fibronectin, ED-B, which is also expressed in the developing neovasculature of tumours, as demonstrated both by immunocytochemistry and by targeting of tumours in vivo. It also relates to materials and methods relating to such specific binding members.
The primary aim of most existing forms of tumour therapy is to kill as many constituent cells of the tumour as possible. The limited success that has been experienced with chemotherapy and radiotherapy relates to the relative lack of specificity of the treatment and the tendency to toxic side-effects on normal tissues. One way that the tumour selectivity of therapy may be improved is to deliver the agent to the tumour through a binding protein, usually comprising a binding domain of an antibody, with specificity for a marker antigen expressed on the surface of the tumour but absent from normal cells. This form of targeted therapy, loosely termed ‘magic bullets’, has been mainly exemplified by monoclonal antibodies (mAbs) from rodents which are specific for so-called tumour-associated antigens expressed on the cell surface. Such mAbs may be either chemically conjugated to the cytotoxic moiety (for example, a toxin or a drug) or may be produced as a recombinant fusion protein, where the genes encoding the mAb and the toxin are linked together and expressed in tandem.
The ‘magic bullet’ approach has had limited, although significant, effect in the treatment of human cancer, most markedly in targeting tumours of lymphoid origin, where the malignant cells are most freely accessible to the therapeutic dose in the circulation. However, the treatment of solid tumours remains a serious clinical problem, in that only a minute proportion of the total cell mass, predominantly the cells at the outermost periphery of the tumour, is exposed to therapeutic immunoconjugates in the circulation; these peripheral targets form a so-called ‘binding site barrier’ to the tumour interior (Juweid et al, 1992, Cancer Res. 52 5144-5153). Within the tumour, the tissue architecture is generally too dense with fibrous stroma and closely packed tumour cells to allow the penetration of molecules in the size range of antibodies. Moreover, tumours are known to have an elevated interstitial pressure owing to the lack of lymphatic drainage, which also impedes the influx of exogenous molecules. For a recent review of the factors affecting the uptake of therapeutic agents into tumours, see Jain, R (1994), Sci. Am. 271 58-65.
Although there are obvious limitations to treating solid tumours through the targeting of tumour-associated antigens, these tumours do have a feature in common which provides an alternative antigenic target for antibody therapy. Once they have grown beyond a certain size, tumours are universally dependent upon an independent blood supply for adequate oxygen and nutrients to sustain growth. If this blood supply can be interfered with or occluded, there is realistic potential to starve thousands of tumour cells in the process. As a tumour develops, it undergoes a switch to an angiogenic phenotype, producing a diverse array of angiogenic factors which act upon neighbouring capillary endothelial cells, inducing them to proliferate and migrate. The structure of these newly-formed blood vessels is highly disorganised, with blind endings and fenestrations leading to increased leakiness, in marked contrast to the ordered structure of capillaries in normal tissue. Induction of angiogenesis is accompanied by the upregulation of expression of certain cell surface antigens, many of which are common to the vasculature of normal tissues. Identifying antigens which are unique to neovasculature of tumours has been the main limiting factor in developing a generic treatment for solid tumours through vascular targeting. The antigen which is the subject of the present invention addresses this problem directly.
During tumour progression, the extracellular matrix of the surrounding tissue is remodeled through two main processes: (1) the proteolytic degradation of extracellular matrix components of normal tissue and (2) the de novo synthesis of extracellular matrix components by both tumour cells and by stromal cells activated by tumour-induced cytokines. These two processes, at steady state, generate a ‘tumoral extracellular matrix’, which provides a more suitable environment for tumour progression and is qualitatively and quantitatively distinct from that of normal tissues. Among the components of this matrix are the large isoforms of tenascin and fibronectin (FN); the description of these proteins as isoforms recognises their extensive structural heterogeneity which is brought about at the transcriptional, post-transcriptional and post-translational level (see below). It is one of the isoforms of fibronectin, the so-called B+ isoform (B-FN), that is the subject of the present invention.
Fibronectins (FN) are multifunctional, high molecular weight glycoprotein constituents of both extracellular matrix and body fluids. They are involved in many different biological processes such as the establishment and maintenance of normal cell morphology, cell migration, haemostasis and thrombosis, wound healing and oncogenic transformation (for reviews see Alitalo et al., 1982; Yamada, 1983; Hynes, 1985; Ruoslahti et al., 1988; Hynes, 1990; Owens et al., 1986). Structural diversity in FNs is brought about by alternative splicing of three regions (ED-A, ED-B and IIICS) of the primary FN transcript (Hynes, 1985; Zardi et al., 1987) to generate at least 20 different isoforms, some of which are differentially expressed in tumour and normal tissue. As well as being regulated in a tissue- and developmentally-specific manner, it is known that the splicing pattern of FN-pre-mRNA is deregulated in transformed cells and in malignancies (Castellani et al., 1986; Borsi et al, 1987; Vartio et al., 1987, Zardi et al, 1987; Barone et al, 1989; Carnemolla et al, 1989; Oyama et al, 1989, 1990; Borsi et al, 1992b). In fact, the FN isoforms containing the ED-A, ED-B and IIICS sequences are expressed to a greater extent in transformed and malignant tumour cells than in normal cells. In particular, the FN isoform containing the ED-B sequence (B+ isoform), is highly expressed in foetal and tumour tissues as well as during wound healing, but restricted in expression in normal adult tissues (Norton et al, 1987; Schwarzbauer et al, 1987; Gutman and Kornblihtt, 1987; Carnemolla et al, 1989; ffrench-Constant et al, 1989; ffrench-Constant and Hynes, 1989; Laitinen et al, 1991.) B+ FN molecules are undetectable in mature vessels, but upregulated in angiogenic blood vessels in normal (e.g. development of the endometrium), pathologic (e.g. in diabetic retinopathy) and tumour development (Castellani et al, 1994).
The ED-B sequence is a complete type III-homology repeat encoded by a single exon and comprising 91 amino acids. In contrast to the alternatively spliced IIICS isoform, which contains a cell type-specific binding site, the biological function of the A+ and B+ isoforms is still a matter of speculation (Humphries et al., 1986).
The presence of B+ isoform itself constitutes a tumour-induced neoantigen, but in addition, ED-B expression exposes a normally cryptic antigen within the type III repeat 7 (preceding ED-B); since this epitope is not exposed in FN molecules lacking ED-B, it follows that ED-B expression induces the expression of neoantigens both directly and indirectly. This cryptic antigenic site forms the target of a monoclonal antibody (mAb) named BC-1 (Carnemolla et al, 1992). The specificity and biological properties of this mAb have been described in EP 0 344 134 B1 and it is obtainable from the hybridoma deposited at the European Collection of Animal Cell Cultures, Porton Down, Salisbury, UK under the number 88042101. The mAb has been successfully used to localise the angiogenic blood vessels of tumours without crossreactivity to mature vascular endothelium, illustrating the potential of FN isoforms for vascular targeting using antibodies.
However, there remain certain caveats to the specificity of the BC-1 mAb. The fact that BC-1 does not directly recognize the B+ isoform has raised the question of whether in some tissues, the epitope recognized by BC-1 could be unmasked without the presence of ED-B and therefore lead indirectly to unwanted crossreactivity of BC-1. Furthermore, BC-1 is strictly specific for the human B+ isoform, meaning that studies in animals on the biodistribution and tumour localisation of BC-1 are not possible. Although polyclonal antibodies to recombinant fusion proteins containing the B+ isoform have been produced (Peters et al, 1995), they are only reactive with FN which has been treated with N-glycanase to unmask the epitope.
A further general problem with the use of mouse monoclonal antibodies is the human.anti-mouse antibody (HAMA) response (Schroff et al, 1985; Dejager et al, 1988). HAMA responses have a range of effects, from neutralisation of the administered antibody leading to a reduced therapeutic dose, through to allergic responses, serum sickness and renal impairment.
Although polyclonal antisera reactive with recombinant ED-B have been identified (see above), the isolation of mAbs with the same specificity as BC-1 following immunisation of mice has generally proved difficult because human and mouse ED-B proteins show virtually 100% sequence homology. The human protein may therefore look like a self-antigen to the mouse which then does not mount an immune response to it. In fact, in over ten years of intensive research in this field, only a single mAb has been identified with indirect reactivity to the B+ FN isoform (BC-1), with none recognising ED-B directly. It is almost certainly significant that the specificity of BC-1 is for a cryptic epitope exposed as a consequence of ED-B, rather than for part of ED-B itself, which is likely to be absent from mouse FN and therefore not seen as “self” by the immune system of the mouse.
Realisation of the present invention has been achieved using an alternative strategy to those previously used and where prior immunisation with fibronectin or ED-B is not required: antibodies with specificity for the ED-6 isoform have been obtained as single chain Fvs (scfvs) from libraries of human antibody variable regions displayed on the surface of filamentous bacteriophage (Nissim et al., 1994; see also WO92/01047, WO92/20791, WO93/06213, WO93/11236, WO93/19172).
We have found using an antibody phage library that specific scFvs can be isolated both by direct selection on recombinant FN-fragments containing the ED-B domain and on recombinant ED-B itself when these antigens are coated onto a solid surface (“panning”). These same sources of antigen have also been successfully used to produce “second generation” scFvs with improved properties relative to the parent clones in a process of “affinity maturation”. We have found that the isolated scFvs react strongly and specifically with the B+ isoform of human FN without prior treatment with N-glycanase.
In anti-tumour applications the human antibody antigen binding domains provided by the present invention have the advantage of not being subject to the HAMA response. Furthermore, as exemplified herein, they are useful in immunohistochemical analysis of tumour tissue, both in vitro and in vivo. These and other uses are discussed further herein and are apparent to the person of ordinary skill in the art.
Terminology
Specific Binding Member
This describes a member of a pair of molecules which have binding specificity for one another. The members of a specific binding pair may be naturally derived or wholly or partially synthetically produced. One member of the pair of molecules has an area on its surface, or a cavity, which specifically binds to and is therefore complementary to a particular spatial and polar organisation of the other member of the pair of molecules. Thus the members of the pair have the property of binding specifically to each other. Examples of types of specific binding pairs are antigen-antibody, biotin-avidin, hormone-hormone receptor, receptor-ligand, enzyme-substrate.
Antibody
This describes an immunoglobulin whether natural or partly or wholly synthetically produced. The term also covers any polypeptide or protein having a binding domain which is, or is homologous to, an antibody binding domain. These can be derived from natural sources, or they may be partly or wholly synthetically produced. Examples of antibodies are the immunoglobulin isotypes and their isotypic subclasses; fragments which comprise an antigen binding domain such as Fab, scFv, Fv, dAb, Fd; and diabodies.
It is possible to take monoclonal and other antibodies and use techniques of recombinant DNA technology to produce other antibodies or chimeric molecules which retain the specificity of the original antibody. Such techniques may involve introducing DNA encoding the immunoglobulin variable region, or the complementarity determining regions (CDRs), of an antibody to the constant regions, or constant regions plus framework regions, of a different immunoglobulin. See, for instance, EP-A-184187, GB 2188638A or EP-A-239400. A hybridoma or other cell producing an antibody may be subject to genetic mutation or other changes, which may or may not alter the binding specificity of antibodies produced.
As antibodies can be modified in a number of ways, the term “antibody” should be construed as covering any specific binding member or substance having a binding domain with the required specificity. Thus, this term covers antibody fragments, derivatives, functional equivalents and homologues of antibodies, including any polypeptide comprising an immunoglobulin binding domain, whether natural or wholly or partially synthetic. Chimeric molecules comprising an immunoglobulin binding domain, or equivalent, fused to another polypeptide are therefore included. Cloning and expression of chimeric antibodies are described in EP-A-0120694 and EP-A-0125023.
It has been shown that fragments of a whole antibody can perform the function of binding antigens. Examples of binding fragments are (i) the Fab fragment consisting of VL, VH, CL and CH1 domains; (ii) the Fd fragment consisting of the VH and CH1 domains; (iii) the Fv fragment consisting of the VL and VH domains of a single antibody; (iv) the dAb fragment (Ward et al., 1989) which consists of a VH domain; (v) isolated CDR regions; (vi) F(ab′)2 fragments, a bivalent fragment comprising two linked Fab fragments (vii) single chain Fv molecules (scFv), wherein a VH domain and a VL domain are linked by a peptide linker which allows the two domains to associate to form an antigen binding site (Bird et al, 1988; Huston et al, 1988) (viii) bispecific single chain Fv dimers (PCT/US92/09965) and (ix) “diabodies”, multivalent or multispecific fragments constructed by gene fusion (WO94/13804; Holliger et al, 1993).
Diabodies are multimers of polypeptides, each polypeptide comprising a first domain comprising a binding region of an immunoglobulin light chain and a second domain comprising a binding region of an immunoglobulin heavy chain, the two domains being linked (e.g. by a peptide linker) but unable to associate with each other to form an antigen binding site: antigen binding sites are formed by the association of the first domain of one polypeptide within the multimer with the second domain of another polypeptide within the multimer (WO94/13804).
Where bispecific antibodies are to be used, these may be conventional bispecific antibodies, which can be manufactured in a variety of ways (Holliger and Winter, 1993), eg prepared chemically or from hybrid hybridomas, or may be any of the bispecific antibody fragments mentioned above. It may be preferable to use scFv dimers or diabodies rather than whole antibodies. Diabodies and scFv can be constructed without an Fc region, using only variable domains, potentially reducing the effects of anti-idiotypic reaction. Other forms of bispecific antibodies include the single chain “Janusins” described in Traunecker et al, (1991).
Bispecific diabodies, as opposed to bispecific whole antibodies, may also be particularly useful because they can be readily constructed and expressed in E. coli. Diabodies (and many other polypeptides such as antibody fragments) of appropriate binding specificities can be readily selected using phage display (WO94/13804) from libraries. If one arm of the diabody is to be kept constant, for instance, with a specificity directed against antigen X, then a library can be made where the other arm is varied and an antibody of appropriate specificity selected.
Antigen Binding Domain
This describes the part of an antibody which comprises the area which specifically binds to and is complementary to part or all of an antigen. Where an antigen is large, an antibody may only bind to a particular part of the antigen, which part is termed an epitope. An antigen binding domain may be provided by one or more antibody variable domains. Preferably, an antigen binding domain comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).
Specific
This refers to the situation in which one member of a specific binding pair will not show any significant binding to molecules other than its specific binding partner. The term is also applicable where eg an antigen binding domain is specific for a particular epitope which is carried by a number of antigens, in which case the specific binding member carrying the antigen binding domain will be able to bind to the various antigens carrying the epitope.
Functionally Equivalent Variant Form
This refers to a molecule (the variant) which although having structural differences to another molecule (the parent) retains some significant homology and also at least some of the biological function of the parent molecule, e.g. the ability to bind a particular antigen or epitope. Variants may be in the form of fragments, derivatives or mutants. A variant, derivative or mutant may be obtained by modification of the parent molecule by the addition, deletion, substitution or insertion of one or more amino acids, or by the linkage of another molecule. These changes may be made at the nucleotide or protein level. For example, the encoded polypeptide may be a Fab fragment which is then linked to an Fc tail from another source. Alternatively, a marker such as an enzyme, flourescein, etc, may be linked.